1. Field of the Invention
The present invention relates to recombinant negative strand RNA molecules which may be used to express heterologous proteins in animal cells and/or to construct recombinant viruses able to express heterologous proteins during their multiplication in host animal cells.
2. Discussion of the Background
Despite the segmented nature of the influenza virus genome, several approaches have already been described to achieve the construction of stable recombinant influenza viruses, able to express heterologous protein sequences of interest. These approaches have been made possible after the development of reverse genetics techniques for the influenza viruses.
Short foreign polypeptides can be expressed by inserting the foreign sequence into an essential viral gene (see, for example, Castrucci, M. R., S. Hou, P. C. Doherty, and Y. Kawaoka 1994. Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes Journal of Virology. 68:3486-90). This strategy is limited to the use of small size epitope inserts and the insertion of a foreign epitope into the sequence of a viral protein can modify the protein in a way which would prevent the generation of viable recombinant viruses
Longer polypeptides can be expressed by the use of a fusion protein containing a protease sensitive peptide sequence between an essential viral gene product and a foreign polypeptide (Percy, N., W. S. Barclay, A. Garcia Sastre, and P. Palese 1994. Expression of a foreign protein by influenza A virus Journal of Virology. 68:4486-4492), but this approach results in the expression of altered viral and/or foreign proteins due to the presence of the specific protease signal in the cleaved protein products.
A foreign polypetide can also be expressed via the use of recombinant RNA segment which were made functionally dicistronic or tricistronic at the level of translation by the insertion of the IRES (Internal Ribosome Entry site) of the human BiP RNA (Garcia-Sastre, A., T. Muster, W. S. Barclay, N. Percy, and P. Palese 1994. Use of a mammalian internal ribosomal entry site element for expression of a foreign protein by a transfectant influenza virus Journal of Virology. 68:6254-6261). In this case, the bicistronic mRNA (which is transcribed during viral replication) permits internal initiation of translation of viral sequences (via internal binding of the ribosome to the IRES sequence) and allows for the expression of foreign protein coding sequences via cap-dependant initiation of translation. A potential drawback of this approach could rely in the fact that the synthesis of the NA gene, which is under the dependance of the IRES sequence is not regulated to the same level and/or at the same time point as it is in the wild type virus ; this could modify the phenotypic characteristics of the virus as it has been shown, for example, that the amount of NA incorporated in the influenza virions affects the infectivity of the viral particles.
Garcia-Sastre et al. (Garcia-Sastre, A., N. Percy, W. Barclay, and P. Palese 1994. Introduction of foreign sequences into the genome of influenza A virus Developments in Biological Standardization. 82:237-246) reported the design of a similar dicistronic RNA molecule in which the corresponding mRNA contains the viral sequence in a proximal position which allows translation from the regular terminal open reading frame, while the translation of the foreign sequence would be initiated from an internal site. In this publication, the IRES of the EMCV virus was used but turned out to be non functional in influenza virus infected cells, since no expression of the foreign sequence could be detected. What is noticeable here is the presence of a duplication of the 39 last nucleotides of NA ORF, which extended the 5xe2x80x2 terminus of the recombinant vRNA molecule from the 28 nt of the 5xe2x80x2 non coding region itself to the 67 first nucleotides of the 5xe2x80x2 terminus of the original NA segment of virus WSN. Nevertheless, this duplication seemed to arise from a facility in the genetic construction of the RNA molecule, since any data neither demonstrates nor shows the role of these additional 39 nt at the 5xe2x80x2 end of the recombinant NA segment encoded by the pT3NA/EMC and pT3NA/EMC-NS 1 plasmids.
More recently, Flick and Hobom (Flick, R., and G. Hobom 1999. Transient bicistronic vRNA segments for indirect selection of recombinant influenza viruses Virology. 262:93-10) introduced another principle of constructing dicistronic vRNA molecules for the transient expression of foreign sequences and/or selection genes. They showed that a recombinant vRNA-like molecule can be made dicistronic by the duplication of its 3xe2x80x2-non coding flanking sequence : in this case, the recombinant RNA molecule is functionally bicistronic for its replication and transcription, in the sense that it can be transcribed and replicated in (a) a genomic mode after interaction of the single 5xe2x80x2 non coding sequence with the external 3xe2x80x2 non coding sequence and in (b) a subgenomic mode after interaction of the 5xe2x80x2 non coding sequence with the internal 3xe2x80x2 non coding sequence. Two types of mRNA molecules are produced, genomic mRNA allows translation of the first cistron, whereas the shorter subgenomic mRNA drives the translation of the second cistron. They showed that the recombinant dicistronic RNA molecules can be propagated as an additional ninth segment during a few rounds of viral multiplication, but that next, the distal segment is spontaneously lost through vRNA-internal initiation events, giving rise to a monocistronic ninth segment. Moreover, it can be anticipated that this resulting virus will be unstable since additional, independant vRNA segments coding for foreign genes are lost during virus growth in the absence of continuing selective pressure as it has been reported by others and the authors themselves in another publication (Neumann, G., and G. Hobom 1995. Mutational analysis of influenza virus promoter elements in vivo Journal of General Virology. 76:1709-17).
Based on the disadvantages of the prior studies, the present inventors sought to design dicistronic influenza genomic segments, in which the first cistron would drive the translation of an essential viral gene and the second cistron would permit the translation of foreign sequences. In completing the invention the present inventors demonstrate that such dicistronic viral segment permitted the rescue of stable recombinant influenza viruses, able to express foreign sequences of interest. The inventors further demonstrate that such recombinant influenza viruses may be used to express heterologous proteins in an animal host. In particular, when inoculated in an animal recipient, these recombinant viruses are able to induce an immune response against the encoded foreign protein. It should be underlined that Flick and Hobom neither reported nor suggested the construction of such dicistronic genomic segments or related recombinant viruses.
The present inventors, for the first time, demonstrate the effectiveness of using recombinant RNA molecules, which were made functionally dicistronic or multicistronic at the level of RNA replication and transcription.
Objects of the Present Invention are as Follows:
A recombinant RNA molecule comprising, from the 3xe2x80x2 end towards the 5xe2x80x2 end:
a) At least two units, each of them composed of a wild-type truncated or mutated 3 xe2x80x2-non coding flanking sequence of a genomic RNA segment of a segmented negative strand RNA virus, optionaly a given spacer sequence of a size chosen from 0 nucleotide to 500 nucleotides, the reverse complement of an mRNA coding sequence or of a fragment of an mRNA coding sequence linked in frame to an initiating AUG and termination codon, a second spacer sequence of a size choosen from 0 nucleotide to 500 nucleotides.
b) A wild-type, truncated or mutated 5xe2x80x2-non coding flanking sequence of a genomic RNA segment of a segmented negative strand RNA virus
A recombinant RNA molecule as described above, wherein the spacer sequence of step A) has preferably a size choosen from 15 to 150 nucleotides.
The recombinant RNA molecule as described above, wherein the first unit, located at the 3xe2x80x2 extremity of the RNA molecule, comprises the reverse complement of the coding sequence selected from the group consisting of a nonmutated viral protein, mutated viral protein, a truncated viral protein, and a chimeric viral protein.
A recombinant virus comprising a segmented negative strand virus, in which at least one genomic segment has been substituted with the recombinant RNA molecule as described above.
The recombinant virus as described above, wherein the first unit, located at the 3xe2x80x2 extremity of the recombinant RNA segment contains the reverse complement of the coding sequence for the viral protein which was encoded by the substituted segment.
The recombinant virus as described above, wherein the virus is a virus choosen among the Orthomyxoviridae, Bunyaviridae or Arenaviridae families.
The recombinant virus as described above, wherein the virus is an influenza virus.
The recombinant virus as described above, in which the substituted genomic segment contains only two units and the second unit comprises the reverse complement of an mRNA coding sequence for an heterologous protein of interest.
The recombinant virus as described above, in which the substituted genomic RNA segment of a segmented negative strand virus is the neuraminidase (NA) segment (segment 6) of influenza virus.
The recombinant virus as described above, in which the 5xe2x80x2-non coding flanking sequences of the recombinant RNA segment are replaced by a longer polynucleotide RNA fragment from the 5xe2x80x2 end of an influenza genomic RNA segment
A purified polynucleotide comprising:
a) a wild-type, truncated or mutated 3xe2x80x2 noncoding flanking viral sequence of a genomic RNA segment of a segmented negative strand RNA virus associated upstream with the reverse complement of a viral Open Reading Frame of a segmented negative strand RNA virus,
b) at least one sequence constitued by a wildtype, truncated or mutated 3xe2x80x2 noncoding flanking sequence of a genomic RNA segment of a segmented negative strand RNA virus associated upstream with a reverse complement Open Reading Frame with a heterologous Open Reading Frame or a polynucleotide of interest, and
c) a wild-type, truncated or mutated 5xe2x80x2 non coding viral sequence.
A purified polynucleotide as described above comprising:
a) a wild-type, truncated or mutated 3xe2x80x2 noncoding flanking viral sequence of a genomic RNA segment of a segmented negative strand RNA virus associated upstream with the reverse complement of a viral Open Reading Frame of a segmented negative strand RNA virus,
b) at least one sequence constituted by a duplication of the same 3xe2x80x2 noncoding flanking sequence associated upstream with the reverse complement of a heterologous gene or a polynucleotide of interest,
c) a wild-type, truncated or mutated 5xe2x80x2 noncoding sequence of the same origin as the 3xe2x80x2 noncoding sequence above.
A recombinant segmented negative strand RNA virus comprising a purified polynucleotide as described above, wherein said purified polynucleotide comprises at least one or more duplication of its 3 xe2x80x2 noncoding flanking sequence with upstream one or more heterologous genes of interest in at least one of its genome segments.
A recombinant segmented negative strand RNA virus comprising a purified polynucleotide as described above which comprises a spacer located upstream the heterologous gene of interest and downstream the 5xe2x80x2 noncoding flanking sequence, wherein said spacer corresponds to at least one or more nucleotide of the genomic RNA segment of a segmented negative strand RNA virus up to the entire sequence, said sequence has been made non coding by the disruption of its Open Reading Frame.
A recombinant virus as described above wherein the virus is an Influenza virus.
A recombinant segmented negative strand virus comprising a purified polynucleotide as described above, wherein viral Open Reading Frame is encoding for the neuraminidase (NA).
A recombinant segmented negative strand virus comprising a purified polynucleotide as described above, wherein the spacer located upstream the heterologous gene of interest and dowstream the 5xe2x80x2 noncoding flanking sequence correspond to at least the reverse complement of the 39 last nucleotides of a coding sequence plus termination codon of a segmented negative strand RNA virus.
A recombinant segmented negative strand virus comprising a purified polynucleotide as described above, wherein the spacer located upstream the heterologous gene of interest and downstream the 5xe2x80x2 noncoding flanking sequence correspond to the reverse complement of the 39 last nucleotides plus termination codon of the neuraminidase gene of said virus.
A purified polypeptide encoded by a polynucleotide as described above and contained in a recombinant virus according to any one of 4 to 10, wherein said polypeptide has the biological characteristic to induce and/or modulate and/or increase the immune response in a host against viral bacterial, fungal or tumoral diseases.
A viral vector useful for delivering an adjuvant of immunity constitued by a recombinant virus as described above.
A viral vector useful for delivering of a biologically active protein of interest comprising the a recombinant virus as described above.
A composition comprising a recombinant virus as described above.
A vaccine composition comprising a recombinant virus as described above.
A method for the induction in the mucosal tissues of a protective response against an infectious agent or a tumoral disease comprising the delivery of a composition as described above.
A method for the induction of a protective response as described above, wherein the mucosal tissue is choosen among the nasal and/or the pulmonary tissues.
A method for producing a recombinant virus, comprising culturing an eucaryotic cell transfected with a vector as described above said cells being infected with a parental strain of a segmented negative strand RNA virus, and recovering the recombinant virus from the resulting culture.
A method as described above, wherein the recombinant virus is a recombinant influenza virus.
A therapeutic composition comprising a recombinant virus as described above.
A kit comprising a composition as described above.
A recombinant virus vNA38-CAT deposited at the C.N.C.M. on Apr. 12, 2001 under the accession number I-2657. A recombinant virus vNA38-S deposited at the CNCM on Apr. 15, 2002 under the accesion number I-2848.
A recombinant DNA molecule corresponding to the recombinant RNA molecule as described above after retrotranscription of said RNA.
A recombinant DNA molecule corresponding to the recombinant viral genome of the recombinant virus as described above after retrotranscription of said RNA.
A process for obtaining the expression in a human or animal host or in a culture of eukaryotic cells of a molecule of interest characterized by infecting said host or culture cells by a recombinant virus as described above.
A composition comprising the recombinant RNA molecule as described above and one or more pharmaceutically acceptable ingredients.
A composition comprising the recombinant segmented negative strand virus as described above and one or more pharmaceutically acceptable ingredients.
A composition comprising the purified polynucleotide as described above and one or more pharmaceutically acceptable ingredients.
A kit comprising the recombinant RNA molecule as described above and one or more reagents for assaying infectivity, immune response (CTL or antibody response), gene expression, or protein levels.
A kit comprising the recombinant segmented negative strand virus as described above and one or more reagents for assaying infectivity, immune response (CTL or antibody response), gene expression, or protein levels.
A kit comprising the purified polynucleotide as described above and one or more reagents for assaying infectivity, immune response (CTL or antibody response), gene expression, or protein levels.
Other objects of the present invention are vaccines comprising the recombinant RNA molecule, the recombinant segmented negative strand RNA virus and/or the purified polynucleotide admixed with one or more adjuvants.
Other objects of the present invention are methods of inducing a protective response against infectious agents or a tumoral disease comprising administering the recombinant RNA molecule,the recombinant segmented negative strand virus and/or the purified polynucleotide to a mucosal tissue. Preferably, the administering further comprises the administering of an adjuvant. In one embodiment of the invention, the a mucosal tissue is nasal mucosal tissue or pulmonary mucosal tissue.
Other objects of the present invention are compositions comprising the recombinant RNA molecules, the recombinant segmented negative strand viruses, and/or the purified polynucleotide and one or more pharmaceutically acceptable ingeredients.
Other objects of the present invention are kits containing the recombinant RNA molecules and/or the recombinant segmented negative strand viruses and/or the purified polynucleotides and one or more reagents for assaying infectivity, immune response, gene expression or protein levels.